Loss or mutation of the p53 tumor suppressor gene is an important event in the malignant transformation of human astrocytes. The loss of wild-type p53 function renders cells more susceptible to genetic instability, predisposing them to neoplastic transformation. However, the specific genetic alterations that occur in astrocytes following the loss of p53 function have not been defined. While rapid and sensitive techniques for the large-scale analysis of gene expression at the mRNA level have been developed, there has been no comparable technique for proteins. mRNA abundance is only one factor that determines how much of a specific protein is present in a cell. Indeed, protein expression levels often show poor correlation with Mrna expression levels. Therefore, it is desirable to obtain information on the proteome level to gain a more complete understanding of a particular process at the molecular level. The aim of the present proposal is to show the feasibility of developing and utilizing new proteomics technology to identify proteins that are altered in cultured postnatal cortical astrocytes during the process of malignant transformation. In the present application, we propose to:l ) Develop and integrate new methods for the extraction and stable isotope and biotin labeling of proteins expressed in p53+/+ and p53-/- mouse cortical astrocytes; 2) Apply differential stable isotope and biotin labeling technology to obtain proteome-wide precise measurements of differences in the protein expression profiles extracted from normal (p53+/+) and malignant mouse astrocytes (p53-/-). Protein expression profiles will be compared with mRNA expression profiles to demonstrate the degree to which changes in mRNA expression are conserved at the protein level, providing insight regarding the extent of post-transcriptional regulation in the process of malignant transformation; and 3) Validate proteome-wide precise measurements of differences in the protein expression profiles extracted from normal (p53+/+) and malignant mouse astrocytes (p53-/-) using immunological and biochemical techniques. The development of extraction protocols and mass spectrometric techniques that can be used to reproducibly measure global changes in protein expression in brain tissue may provide unique insights into the process of neoplastic transformation and may provide new targets for suppressing the growth of human brain tumors.